High affinity cooperative DNA binding by the yeast Mlh1-Pms1 heterodimer

We demonstrate here that the Saccharomyces cerevisiae Mlh1-Pms1 heterodimer required for DNA mismatch repair and other cellular processes is a DNA bin ding protein. Binding was evaluated using a variety of single and double-st randed DNA molecules. Mlh1-Pms1 bound short substrates with low affinity an d showed a slight preference for single-stranded DNA. In contrast, Mlh1-Pms 1 exhibited a much higher affinity for long DNA molecules, suggesting that binding is cooperative. High affinity binding required a duplex DNA length greater than 241 base-pairs. The rate of association with DNA was rapid and dissociation of protein-DNA complexes following extensive dilution was ver y slow. However, in competition experiments, we observed a rapid active tra nsfer of Mlh1-Pms1 from labeled to unlabeled DNA. Binding was non-sequence specific and highly sensitive to salt type and concentration, suggesting th at Mlh1-Pms1 primarily interacts with the DNA backbone via ionic contacts. Cooperative binding was observed visually by atomic force microscopy as lon g, continuous tracts of Mlh1-Pms1 protein bound to duplex DNA. These images also showed that Mlh1-Pms1 simultaneously interacts with two different reg ions of duplex DNA. Taken together, the atomic force microscope images and DNA binding assays provide strong evidence that Mlh1-Pms1 binds duplex DNA with positive cooperativity and that there is more than one DNA binding sit e on the heterodimer. These DNA binding properties of Mlh1-Pms1 may be rele vant to its participation in DNA mismatch repair, recombination and cellula r responses to DNA damage.